Bacterial endotoxins are potent pyrogens that often produces fever reactions when administered to patients. The endotoxin is an integral component of the outer cell membrane of Gram negative bacteria. It exists in its natural stage as a complex of lipid, carbohydrate and protein.
The outer membrane of Gram negative bacteria serves as an outer barrier through which materials must penetrate, if they are to reach the cell. It is selectively permeable. Grown in vitro Gram negative bacteria often release large amounts of endotoxins, up to approximately 1000 mg/ml.
Fermentation has long been a key technology for mass production of products in Gram negative bacteria. An economic fermentation process usually has to demonstrate an optimal cell production, maximum accumulation of the desired product and minimum consumption of nutrients. In most industrial applications, fed-batch cultures have been found preferable over batch cultures due to that the fed-batch technique provides means to obtain higher productivity and higher product concentration compared to batch cultures. This is achieved by controlling the nutrient feeding to regulate the biological reaction rates like e.g. those of energy metabolism (respiration) and growth in a desired range. It is necessary to control the energy metabolism to avoid oxygen limitation in the culture. Controlled feeding of the energy/carbon source is also an effective method to avoid acetic acid accumulation due to overflow metabolism. Production of acetic acid reduces the yield of biomass and the desired product or products.
However, regulation performed solely by the nutrient feeding, usually by a carbon/energy source such as glucose, results in an excessive release of endotoxins into the medium in which the Gram negative bacteria is cultivated. The release of endotoxins results in the need of several purification steps to eliminate the undesired endotoxins from the product prior to use, when the product is to be administered to mammals (e.g. humans).
Endotoxins in large quantities can cause shock, severe diarrhea, fever and leuopenia followed by leukocytosis, elicit the Schwartzman and Sanarelli-Schwartzman phenomena and in severe cases, death of recipient patients. Therefor it is highly important to eliminate the endotoxins prior to use.
A number of methods for removal of endotoxins are known in the art, such as rinsing with nonpyrogenic solution (Feldstine et al., 1979, J. Peranter. Drug. Assoc., 33:12), distillation, ultrafiltration using membranes rated by molecular weight exclusion (Sweadner et al., 1977, Appl. Environ. Microbiol., 34:382, reverse osmosis using thin cellulose acetate or polyamide materials (Neslon, 1978, Pharm. Technol., 2:46), electrostatic attraction (Gerba et al., 1980, Pharm Technom., 4:83), hydrophobic attraction using aliphatic polymers (Robinson et al., 1985; Parental Drug Association, 54–69), adsorption using activated carbon (Berger et al., 1956, dv. Chem. Ser., 16:169) and affinity chromatography (Soter, 1984, Bio/Technol, 12:1035) among others.
The purification steps presently available for the removal of endotoxins are expensive and several steps are necessary for the satisfactory removal of the endotoxins. During the removal of the endotoxins, all the purification steps results in a reduced amount of the biological product of interest as well as the endotoxins.
The invention provides an improved method to be used for the cultivation of bacteria such as Gram negative bacteria without substantial release of endotoxins and without substantial formation of acetic acid. Thereby the need of purification steps for removal of the endotoxins is reduced or eliminated and still the biomass yield is high as well as the accumulation of the desired produced product.